This protocol demonstrate protein crystallization on a chip-like device in x-ray diffraction data recollection. This device is called a crystal-on-crystal because protein crystals grow on a single quartz crystal. Upon successful growth of protein crystals on such devices, thousands of diffraction images are collected from each device at room temperature without even touching the protein crystals.
X-ray diffraction at room temperature is very essential for studying protein function involving many conformational states. This informative protein changes or protein actions can be frozen, thus not detectable in cryo-crystallography. This devices can be used to study light-induced signaling processes and redox changes.
Only a handful of devices gives complete and redundant datasets. To begin device pre-assembly, label the outer ring for the sample identification. Include the project name, device number, crystallization condition, and date as desired.
Then place the labeled ring upside down on a clean surface and place one quartz wafer inside the outer ring. Next, pour a small amount of microscope immersion oil into a Petri dish and dip a shim in the oil ensuring that both sides of the shim are well coated. Remove any excess oil by dabbing the shim on a clean surface.
Then place the oiled shim on top of the first quartz wafer. Mix the protein solution and crystallization buffer on the first quartz wafer using a pipette. Try to avoid air bubbles when mixing.
The total volume of the crystallization solution should not exceed the maximum capacity of the crystallization chamber determined by the shim size and thickness. Place the second quartz wafer over the mixed solution. The solution will spread out spontaneously.
Then tap the second quartz wafer lightly on the edge to help spread the oil while pushing air out. Secure the device by screwing a retaining ring into the outer ring. If necessary, use a tightening tool.
Avoid over-tightening as it may cause delicate quartz wafers to deform or crack. Store the assembled devices in a box at room temperature or inside a temperature-controlled incubator. After a few hours or days, place the crystallization device under a microscope and monitor the crystal growth.
If necessary, optimize crystallization conditions as described in the manuscript. For calibration, install a thin YAG crystal on the chip holder, then install the beam stop. Next, open the Insitux software and run the indicated program to take x-ray fluorescence images of the direct beam where the device is a user-selected name for the crystallization device and device.
param is the filename that contains device-specific control parameters. Then find the precise position of the direct x-ray beam by running the beam profile fitting program where the burn image is the filename of the x-ray fluorescence image. For optical scanning, place a crystallization device in the chip holder and secure using a thumbscrew.
Then mount the chip holder onto the translation stage of the diffractometer through a kinematic mechanism. Depending on the light sensitivity of the protein sample and the purpose of the experiment, install a white or infrared light source to take micrographs from the optical window of the device. Once the setup is ready, run the scan program by entering the indicated command for scanning in motion at the beam line.
Then run the tiling program on a user computer where x is the initial value for the column and y is the initial value for rogue displacements of micrographs. The program stitches all micrographs into a montage of one to three micrometer pixel resolution. After stitching the micrographs, enter the indicated command to run the crystal finding program.
This program performs crystal recognition and shot planning and the key parameters in this program enables specific crystal selection and target planning. Remove the light source and position the beam stop. Then run the data collection program for serial diffraction.
The suggested command triggers data collection by visiting the planned shots in a pre-programmed sequence. Each targeted crystal is translocated to the beam position. At each stop, x-ray exposure is taken either with or without a laser illumination at a scheduled time delay.
In the study, the crystallization conditions between vapor diffusion and an on-chip batch were compared. Four case studies of on-chip crystallization and representative datasets collected directly from quartz devices are demonstrated here. The dynamic crystallography experiments revealed light-induced changes in far red photoreceptor protein by comparing data from 4, 352 crystals in the dark and 8, 287 crystals after light illumination.
The dark dataset from in situ serial Laue diffraction resulted in better resolved electron densities, enabling confident model building of a bilin chromophore in an all Z sign conformation. The light-induced difference maps have revealed concerted motions in the central beta sheet suggesting the importance of the pi-pi stacking between the pyrrole rings of the chromophore and several aromatic residues. This platform is called Insitux.
The unique advantage of this platform is that one doesn't have to do any crystal manipulation once the crystallization is done. It is necessary to collect many datasets under changing conditions to capture the protein motions. And this becomes possible with Insitux because it enables large-scale data collection from thousands of protein crystals at room temperature.
With this new capability including crystallography, the light sensitive systems can be triggered inside the device and the light inert system can be studied if the crystal-on-crystal device is converted into outflows.
